Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds, The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site, Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein, In this study, using [I-125]iodoarylazidoprazosin ([I-125]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [I-125]IAAP binding to the Nand C-terminal halves, cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [I-125]IAAP for the C-terminal half of the protein (C-site) by reducing the K-d from 20 to 6 nM without changing the labeling or affinity (K-d = 42-46 nM) of the N-terminal half (N-site), Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [I-125]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site, In addition, [I-125]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport, These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.